Melanin pigments are responsible for pigmentation of skin hair and eyes and are synthesized in an organelle called melanosome in melanocytes and retinal pigmented epithelial cells. In the case of skin melanocytes, melanin pigments are then transferred to neighboring keratinocytes. Examples of pigmentation disorders characterized by defective melanosome biogenesis and function include Hermansky-Pudlak syndrome, oculocutaneous albinism and Griscelli syndrome. Further, the sequestration and secretion of intracellular cytotoxic drugs by melanosomes underlies the development of multidrug resistance in melanomas. Consequently, inhibiting melanosome function constitutes an important approach to enhance the chemosensitivity of melanoma cells. Thus, understanding the molecular mechanisms regulating pigmentation has physiological significance and will likely lead to clinical applications. In spite of the relevance of pigmentation for human health, there are significant knowledge gaps in our understanding of melanosome biogenesis and function. Our goal is to answer fundamental outstanding questions in this area of the pigmentation field in an integrated manner and focusing on molecular mechanisms. Aim 1: The enzymes tyrosinase, tyrosinase-related protein 1 (Tyrp1) and Tyrp2 synthesize melanin in the melanosome lumen. Melanosome biogenesis requires the delivery of the three melanogenic enzymes to the maturing organelle. The transport of newly synthesized tyrosinase and Tyrp1 to melanosomes is relatively well understood. In contrast, the pathway followed by Tyrp2 to melanosomes is unknown. We have evidence that Tyrp2 uses a different transport route from the one used by tyrosinase and Tyrp1. We will utilize a novel assay we recently developed to define the pathway followed by Tyrp2 and compare it with the pathway used by tyrosinase and Tyrp1. The biogenesis of melanosomes (and other lysosome-related organelles) depends on the function of several proteins that are encoded by genes mutated in various forms of Hermanky-Pudlak syndrome (HPS). The two most common and severe forms of the disease are HPS1 and HPS4, yet little is known about the function of these HPS proteins in organelle biogenesis. We will test the hypothesis that HPS1 and HPS4 together with the small GTPase Rab32 define a transport pathway to melanosomes that is used by Tyrp2 and other melanosomal proteins. Aim 2: A genome-wide association study identified Two-Pore Channel 2 (TPC2) as a genetic determinant of pigmentation but how TPC2 regulates pigmentation is a mystery. Our data suggests TPC2 is a component of the melanosome membrane that regulates melanosome function. We will test the hypothesis that through changes in melanosome pH, TPC2 controls the activity of the tyrosinase enzymes and the amount and type of melanin synthesized. Moreover, we will study the mechanism of TPC2 function and polymorphisms associated with pigmentation variations. Aim 3: After melanosome biogenesis and melanin synthesis take place, skin melanocytes transfer the pigments to keratinocytes. The mechanism for intercellular melanin transfer is unclear but four models have been proposed. We will test which one of the models is correct or if melanin transfer occurs by more than one mechanism. Further, the molecular machinery involved in this process will be investigated.